Understanding the regulatory networks that direct stem cell differentiation is critical to realizing the promise of stem cell therapy. Differentiation requires large and rapid shifts in the mRNA and protein constitution of cells. Small RNAs are particularly well suited to perform this task. For example, microRNAs, a subclass of small RNAs, are predicted to individually regulate hundreds of target genes simultaneously. The overall goal of this proposal is to systematically study the role of small RNAs in embryonic and neural stem cell differentiation. This is a collaborative grant including experts in stem cells and small RNAs. To address the overall objective of the grant, a mouse embryonic stem cell model was developed that specifically knocks-out microRNA function thereby separating the roles of the two main classes of small RNAs, microRNAs and siRNAs, in mammalian development. This model is based on the conditional deletion of the RNA binding protein DGCR8, which is required for the processing of pri- to pre-microRNAs. The specific aims of this grant include: 1) the analysis of role of microRNAs in stem cell proliferation based on preliminary results showing that DGCR8 null ES cells accumulate in the G1 phase of the cell cycle, 2) characterization of the role of miRNAs in stem cell differentiation based on initial findings that DGCR8 null ES cells continue to self-renew even under strict differentiation conditions. 3) identification of novel non-microRNA small RNAs that are required for stem cell function. This aim is based on the finding that the DGCR8 null ES cell phenotype differs from that of the Dicer null phenotype. Completion of this project will help identify regulatory networks that can be manipulated to differentiate and potentially de-differentiate cells along specific developmental pathways. Small RNAs are a relatively new class of cell regulators whose pervasiveness and global effects on gene function suggests they have a central role in both normal and abnormal human development. In support of this, recent evidence shows they have an important role in the development of tumors. Therefore, a systematic analysis of their function in embryonic and somatic stem cells as proposed in this grant should provide important insights into how they influence and can be used to diagnose and treat human disease.